METHOD FOR PREPARING A COMPOSITION COMPRISING AN UNFOLDED PROTEIN

Abstract

The invention provides a substantially water-free composition for use in preparing a biologically active complex, methods for preparing such compositions and to uses thereof.

Claims

1. A method for preparing a composition, the method comprising the steps of combining: a. a dried polypeptide, b. a fatty acid or lipid, or a pharmaceutically acceptable salt thereof, and c. a buffer component comprising at least two salts, preferably wherein the first salt is sodium or potassium chloride and the second salt is disodium phosphate or mono-potassium phosphate, wherein the composition is substantially water-free.

2. A method according to claim 1 wherein the buffer component further comprises a third salt which is mono-sodium or mono-potassium phosphate, preferably mono-potassium phosphate.

3. A method according to any preceding claim, wherein the polypeptide is a membrane-perturbing protein.

4. A method according to any preceding claim, wherein the polypeptide is selected from natural alpha-lactalbumin, SAR1, or lysozyme, or active fragments or active variants thereof.

5. A method according to claim 4, wherein the alpha-lactalbumin is human alpha-lactalbumin or bovine alpha-lactalbumin.

6. A method according to any preceding claim, wherein the active fragment or active variant comprises a polypeptide which lacks intra-molecular bonds.

7. A method according to any preceding claim, wherein the active fragment or active variant is a fragment or variant of up to 50 amino acids.

8. A method according to any preceding claim, wherein the active fragment comprises a polypeptide of any of SEQ ID NOs 5 to 11, preferably a polypeptide of SEQ ID NO 6.

9. A method according to any preceding claim, wherein the first salt is sodium chloride and the second salt is disodium monophosphate.

10. A method according to any preceding claim, wherein the fatty acid is an unsaturated fatty acid, preferably a C18 unsaturated fatty acid, more preferably a cis C18:1:9 or C18:1:11 fatty acid, yet more preferably oleic acid, or a pharmaceutically acceptable salt thereof.

11. A composition obtainable by a method according to any one of the preceding claims.

12. A composition that is substantially water-free comprising: a. a dried polypeptide, b. a fatty acid or a lipid, or a pharmaceutically acceptable salt thereof, and c. a buffer component comprising at least two salts, preferably wherein the first of which is sodium or potassium chloride and the second of which is disodium phosphate or mono-potassium phosphate, wherein the composition is substantially free of polypeptide that is in a complex with the fatty acid or a pharmaceutically acceptable salt thereof.

13. A composition according to claim 12, the composition further comprising the features of any of claims 2 to 10.

14. A method for preparing a biologically active complex, the method comprising the steps of providing a composition according to any of claims 11 to 13, adding an aqueous carrier, and agitating the mixture.

15. A method according to claim 14 wherein the agitation is carried out at a moderate temperature, preferably from 10-40° C., more preferably at ambient temperature.

16. A biologically active complex obtainable by any of claims 14 to 15.

17. A biologically active complex obtainable by any of claims 14 to 15, or the composition of any of claims 11 to 13, for use in therapy.

18. A biologically active complex or composition for use according to claim 17, wherein the use is treatment or prevention of cancer or viral infection.

19. A pharmaceutical or nutraceutical composition comprising a composition according to claims 11 to 13 or a biologically active complex of claim 16.

20. A method for treating or preventing cancer or a viral infection, said method comprising administering to a patient in need thereof an effective amount of a biologically active complex according to claim 16 or a composition according to any of claims 11 to 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The invention will now be particularly described by way of example with reference to the accompanying drawings in which

[0084] FIG. 1 shows a schematic of a composition according to the invention and a method of preparing a biologically active complex by adding sterile water and shaking.

[0085] FIG. 2 shows the results of ATP Lite and PrestoBlue studies obtained using a biologically active complex prepared according to the schematic of FIG. 1 and a control biologically active complex on tumour cells as described below.

EXAMPLE 1

[0086] Production of Biologically Acceptable Complexes

[0087] A composition according to the invention was prepared using a peptide of SEQ ID NO 6, which is a variant of a fragment of human α-lactalbumin.

[0088] As shown in FIG. 1, the peptide (1.7 mM), in lyophilised form, was added to a tube together with pure oleic acid (8.5 mM) and NaCl (6.8 g/L), Na.sub.2HPO.sub.4×2H.sub.2O (4.8 g/L); and KH.sub.2PO.sub.4 (1.3 g/L). The concentrations provided are with respect to the concentration once made up with an appropriate quantity of water. The tube was then mixed by hand.

[0089] To prepare the biologically active complex, sterile water was added to the tube and shaken by hand for 30 to 60 seconds. The result was a clear solution, indicating that the biologically active complex had been formed without any precipitation of protein.

EXAMPLE 2

[0090] Cell Death Assay

[0091] Human lung carcinoma cells (A549, ATCC) were cultured in RPMI-1640 with non-essential amino acids (1:100), 1 mM sodium pyruvate, 50 μg/ml Gentamicin and 5-10% fetal calf serum (FCS) at 37° C., 5% CO.sub.2. For cell death experiment, cells were grown on 96-well plate (2×10.sup.4/well, Tecan Group Ltd) overnight. Cells were incubated with biologically active complexes obtained in Example 1 at dosages equivalent to either 7, 21 or 35 μM peptide in serum-free RPMI-1640 at 37° C. FCS was added after 1 hour. Cell death was quantified 3 hours after peptide-oleate treatment by two biochemical methods including 1) estimation of cellular ATP levels using luminescence based ATPlite™ kit (Perkin Elmer) and 2) Presto Blue fluorescence staining (Invitrogen, A13262). Fluorescence and luminescence was measured using a microplate reader (Infinite F200, Tecan).

[0092] The results are shown in FIG. 2. FIG. 2 shows a comparison of tumour cell death activity of biologically active complex prepared according to the invention with biologically active complex prepared according to the technique of WO 2018/210759. The results show comparable activity, indicating that the composition of the invention is fully capable of being used to form high levels of biologically active complex.